Probiotic compositions and methods

ABSTRACT

The present application relates to compositions and methods for modulation of the mucosal immune system. The compositions include foods fermented by the probiotic organism,  Lactobacillus paracasei  CBA L74 (International Depository Accession Number LMG P-24778). Alternatively, the compositions can include  L. paracasei  CBA L74 and a physiologically acceptable carrier. In some embodiments, the  L. paracasei  CBA L74 can be non-replicating. The compositions can be administered to a subject having or at risk for gastrointestinal disorders related to immaturity of the immune system, infection or disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/393,904, filed Dec. 29, 2016, which is a divisional of U.S.application Ser. No. 14/127,573, filed Dec. 19, 2013, now U.S. Pat. No.10,039,296, which is a national phase application of InternationalApplication No. PCT/US2012/042959, filed Jun. 18, 2012, which claimspriority to U.S. Provisional Application No. 61/498,910, filed Jun. 20,2011, which are incorporated by reference herein in their entireties.

FIELD OF THE INVENTION

The present invention relates to probiotic organisms, food productsprepared with probiotic organisms and pharmaceutical compositionscomprising probiotic organisms. These compositions are useful instimulating the mucosal immune system and for treatment of disordersassociated with immaturity of the mucosal immune system.

BACKGROUND OF THE INVENTION

The intestinal epithelium is constantly exposed to foreign materialsthat can be either harmful or beneficial to the host. As a result, theintestinal immune system must strike a delicate balance between: 1)protective immune responses that are induced by intestinal pathogens ortoxins and 2) avoidance of immune responses against both food antigensand the 10¹⁴ commensal beneficial microorganisms that normally reside inthe gut. Disruption of either the protective responses or the toleranceresponses can result in a wide array of disorders including, forexample, infections, inflammation, food allergies, foodhypersensitivity, inflammatory bowel disease, Crohn's disease, celiacdisease, periodontal disease, rheumatoid arthritis, atherosclerosis andcolon cancer.

The immunoregulatory network comprising the intestinal immune systemchanges with age. The network is poorly developed in human newborns andis established gradually over the first few years of life. Theimmaturity of the immune system plays a role in the prevalence ofinfections and food-related disorders in infants and young children.Conversely, the ability of the intestinal immune system to respond tonew challenges declines in the elderly.

Gastrointestinal disorders, for example, infections, inflammatorydisorders and food-related disorders such as food allergies, foodintolerance or food hypersensitivity have a significant impact on thehealth and quality of life in both children and adults. Infectiousgastroenteritis is the most common pediatric gastrointestinal disorder.About 1 billion episodes occur worldwide each year, most commonly indeveloping countries among children under 5 years of age. Worldwidedeath rates for infectious gastroenteritis average from 3 to 6 millionchildren per year. In the United States, 25 to 35 million new casesoccur annually, resulting in 300 to 400 deaths. In addition, infectiousgastroenteritis in the US results in an estimated 200,000hospitalizations and 1.5 million outpatient visits at a cost in excessof 1 billion dollars. Food-related disorders such as allergies also havea substantial effect on health or both children and adults. Symptoms offood allergies can vary depending upon the severity of the allergy andcan range from a mild tingling sensation around the mouth and lips tolife-threatening anaphylaxis. It is estimated that food allergies affectbetween 1-10% of the population in the U.S. The Center for DiseaseControl found that in 2007, approximately 3 million children under age18 years (3.9%) were reported to have a food or digestive allergy in theprevious 12 months. For some children, food allergies become less severewith age, for others, they remain a lifelong concern. Infants who sufferfrom allergy early in life may develop “allergic march.” For example,many individuals who have severe allergic reactions to cow's milk ininfancy at risk for the development of asthma later in childhood. Thereare indications that the prevalence of food allergies is increasingworldwide.

Regardless of the etiology, gastrointestinal disorders not onlyadversely affect a child's health, but can have a serious impact onfamily economics, social interactions and school and parental workattendance. There is a continuing need for therapeutic strategies thatpromote gastrointestinal health, particularly in individuals who arerisk for or who suffer from gastrointestinal disorders.

SUMMARY OF THE INVENTION

The present invention provides compositions comprising a fermented foodproduct, wherein the food product has been fermented by the probioticbacterium, Lactobacillus paracasei CBA L74, International DepositoryAccession Number LMG P-24778. The food product can be a dairy product ora cereal product. Also provided are compositions comprising theprobiotic bacterium, Lactobacillus paracasei CBA L74, InternationalDepository Accession Number LMG P-24778 and a physiologically acceptablecarrier. The physiologically acceptable carrier can be a food product ora pharmaceutical carrier. Also provided are methods of making anutritional composition, the method comprising: providing a foodproduct; combining the food product with an effective amount of theprobiotic bacterium, Lactobacillus paracasei CBA L74, InternationalDepository Accession Number LMG P-24778 and, optionally, a co-inoculum,to form a mixture; and incubating the mixture at a temperature and for atime sufficient for fermentation to occur. The nutritional compositionmay be dried. The nutritional composition may be combined with one ormore additional food products. For any of the compositions and methodsdescribed herein, the Lactobacillus paracasei CBA L74 cells can besubjected to treatments that render them non-replicating. Theconcentration of Lactobacillus paracasei CBA L74 in the compositions canvary depending upon the intended use, e.g., as a nutritional compositionor a pharmaceutical composition. Useful ranges include the equivalent ofabout 1×10² colony-forming units per gram (“cfu/g”) to about 1×10¹²colony-forming units per gram (“cfu/g”) dry weight.

Also provided are methods of treating a subject at risk for a developinga gastrointestinal disorder, the method comprising: identifying asubject at risk for a gastrointestinal disorder; administering aneffective amount of a composition comprising a food product wherein thefood product has been fermented by the probiotic bacterium,Lactobacillus paracasei CBA L74, International Depository AccessionNumber LMG P-24778. The gastrointestinal disorder can be a mucosalimmune system deficit, for examine, an immature immune system, a foodallergy, a disorder associated with diarrhea, a bacterial or viralinfection, irritable bowel syndrome, inflammatory bowel disease, Crohn'sdisease, necrotizing enterocolitis or aging, particularly aging of thegastrointestinal system.

Also provided are methods of treating a subject having agastrointestinal disorder. The methods include: identifying a subjecthaving a gastrointestinal disorder; administering an effective amount ofa composition comprising a food product wherein the food product hasbeen fermented by the probiotic bacterium, Lactobacillus paracasei CBAL74, International Depository Accession Number LMG P-24778. In someembodiments, the methods include: identifying a subject having agastrointestinal disorder; administering an effective amount of apharmaceutical composition comprising the probiotic bacterium,Lactobacillus paracasei CBA L74, International Depository AccessionNumber LMG P-24778. The gastrointestinal disorder can be a mucosalimmune system deficit, for examine, an immature immune system, a foodallergy, a disorder associated with diarrhea, a bacterial or viralinfection, irritable bowel syndrome, inflammatory bowel disease, Crohn'sdisease or necrotizing enterocolitis.

Also provided are and methods of modulating the immune system in asubject. The methods include: identifying a subject in need of immunesystem modulation and administering an effective amount of a compositioncomprising a food product wherein the food product has been fermented bythe probiotic bacterium, Lactobacillus paracasei CBA L74, InternationalDepository Accession Number LMG P-24778.

Articles of manufacture are also provided. These can include kitscomprising a measured amount of a nutritional composition comprising afermented food product, wherein the food product has been fermented bythe probiotic bacterium, Lactobacillus paracasei CBA L74, InternationalDepository Accession Number LMG P-24778 and one or more items selectedfrom the group consisting of packaging material, a package insertcomprising instructions for use, a sterile fluid, and a sterilecontainer. In some embodiments, the kit can include a measured amount ofa pharmaceutical composition comprising Lactobacillus paracasei CBA L74,International Depository Accession Number LMG P-24778 and one or moreitems selected from the group consisting of packaging material, apackage insert comprising instructions for use, a sterile fluid, and asterile container.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects and advantages of the invention will be apparent from thedescription and drawings and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features and advantages of the present invention will bemore fully disclosed in, or rendered obvious by, the following detaileddescription of the preferred embodiment of the invention, which is to beconsidered together with the accompanying drawings wherein like numbersrefer to like parts and further wherein:

FIG. 1 is a table showing an analysis of the effect of L. paracasei CBAL74 on DC cell phenotype.

FIG. 2A is a graph depicting IL-10 production in DCs co-cultured withCaco2 exposed to L. paracasei CBA L74. FIG. 2B is a graph depictingIL-12 production in DCs co-cultured with Caco2 exposed to L. paracaseiCBA L74.

FIG. 3 is a graph depicting proliferation of T cells exposed to DCscocultured with CaCo2 cells.

FIG. 4 is a graph depicting IL-1β production in intestinal mucosa ofmice supplemented L. paracasei CBA L74.

FIG. 5 is a graph depicting IL-4 production in intestinal mucosa of micesupplemented with L. paracasei CBA L74.

FIG. 6 is a graph depicting IgA production in intestinal mucosa of micesupplemented with L. paracasei CBA L74.

FIG. 7 depicts an analysis of levels of TLR2, TLR4 and TLR9 inintestinal mucosa of mice supplemented with L. paracasei CBA L74.

FIG. 8 depicts an analysis of levels of PPARγ in intestinal mucosa ofmice supplemented with L. paracasei CBA L74.

FIG. 9A is a graph depicting serum IL-1β levels in mice supplementedwith L. paracasei CBA L74. FIG. 9B is a graph depicting serum IL-4levels in mice supplemented with L. paracasei CBA L74.

FIG. 10 is a table showing an analysis of the effect of L. paracasei CBAL74 on DC phenotype in mice supplemented with L. paracasei CBA L74.

FIG. 11A is a graph depicting intestinal CD4+ lymphocyte phenotypes inmice supplemented with L. paracasei CBA L74. FIG. 11B is a graphdepicting intestinal CD8+ lymphocyte phenotypes in mice supplementedwith L. paracasei CBA L74.

FIG. 12 is a graph depicting IL-10 production in intestinal mucosa ofmice supplemented with milk fermented by L. paracasei CBA L74.

FIG. 13 is a graph depicting IL-1β production in intestinal mucosa ofmice supplemented with milk fermented by L paracasei CBA L74.

FIG. 14 is a graph depicting IgA production in intestinal mucosa of micesupplemented with milk fermented by L. paracasei CBA L74.

FIG. 15A and FIG. 15B each depict an analysis of levels of TLR2, TLR4,TLR9 and PPARγ in intestinal mucosa of mice supplemented with milkfermented by L. paracasei CBA L74.

FIG. 16 depicts an analysis of levels of pNF-kB and IKBα in intestinalmucosa of mice supplemented with milk fermented by L. paracasei CBA.

FIG. 17 depicts an analysis of the effect of L. paracasei CBA L74 on DCcell phenotype in mice supplemented with milk fermented by L. paracaseiCBA.

FIG. 18 is an analysis of the effect of L paracasei CBA L74 on DC cellphenotype after exposure to LPS or CpG in mice supplemented with milkfermented by L. paracasei CBA.

FIG. 19 is a graph depicting intestinal CD4+ lymphocyte phenotypes inmice supplemented with milk fermented by L. paracasei CBA L74.

FIG. 20 is a graph depicting intestinal CD8+ lymphocyte phenotypes inmice supplemented with milk fermented by L. paracasei CBA L74.

FIG. 21 shows histological evaluation of ileal mucosa in micesupplemented with milk fermented by L. paracasei CBA L74.

FIG. 22 is a graph depicting IL-1β and IL-4 production in intestinalmucosa of mice supplemented with rice fermented by L. paracasei CBA L74.

FIG. 23 is a graph depicting IL-10 production in intestinal mucosa ofmice supplemented with rice fermented by L. paracasei CBA L74.

FIG. 24 is an analysis of levels of TLR2 and TLR4 in intestinal mucosaof mice supplemented with rice fermented by L. paracasei CBA L74.

FIG. 25A is an analysis of levels of PPARγ in intestinal mucosa of micesupplemented with rice fermented by L. paracasei CBA L74. FIG. 25B is ananalysis of levels of pNF-kB and IKBα in intestinal mucosa of micesupplemented with rice fermented by L. paracasei CBA L74.

FIG. 26 is a table showing an analysis of the effect of L. paracasei CBAL74 on DC phenotype in mice supplemented with rice fermented by L.paracasei CBA L74.

FIG. 27 is a table showing an analysis of the effect of L. paracasei CBAL74 on DC phenotype after exposure to LPS or CpG in mice supplementedwith rice fermented by L. paracasei CBA L74.

FIG. 28 is a graph depicting intestinal CD4+ lymphocyte phenotypes inmice supplemented with rice fermented by L. paracasei CBA L74.

FIG. 29 is a graph depicting intestinal CD8+ lymphocyte phenotypes inmice supplemented with rice fermented by L. paracasei CBA L74.

FIG. 30 is a graph depicting an analysis of the effect of L. paracaseiCBA L74 cells and cell supernatant on IL-10 production in human MoDCspresence of Salmonella typhimurium.

FIG. 31 is a graph depicting an analysis of the effect of L. paracaseiCBA L74 cells and cell supernatant on IL-12p70 production in human MoDCsin the presence of Salmonella typhimurium.

FIG. 32 is a graph depicting an analysis of the effect of L. paracaseiCBA L74 fermented milk on IL-10 production in human MoDCs presence ofSalmonella typhimurium and the effect of inactivation of L. paracaseiCBA L74 on IL-10 production in human MoDCs in the presence of Salmonellatyphimurium.

FIG. 33 is a graph depicting an analysis of the effect of L. paracaseiCBA L74 fermented milk on IL-12p70 production in human MoDCs presence ofSalmonella typhimurium and the effect of inactivation of L. paracaseiCBA L74 on I L-12p70 production in human MoDCs in the presence ofSalmonella typhimurium.

FIG. 34 is a graph depicting an analysis of the effect of L. paracaseiCBA L74 fermented rice on IL-1β, TNF-α and IL-10 in a gut tissue explantmodel in the presence of Salmonella typhimurium.

DETAILED DESCRIPTION

The present invention is based, in part, on the inventors' discoverythat foods fermented by the probiotic organism Lactobacillus paracasei,strain CBA L74, can have immunomodulatory properties. This strain wasisolated by the inventors and deposited under the Budapest Treaty on theInternational Recognition of the Deposit of Micro-organisms for thePurposes of Patent Procedure on Sep. 9, 2008 at the Belgian CoordinatedCollections of Micro-organisms (BCCM) Laboratorium voor Microbiologie(LMG), Ghent, Belgium. The Accession Number given by the InternationalDepositary Authority is LMG P-24778. For ease of reading, we will notrepeat the phrase “Accession Number LMG P-24778” on every occasion. Itis to be understood that where we refer to L. paracasei, strain CBA L74,we refer to the deposited strain having the Accession Number LMGP-24778.

The compositions of the invention include the probiotic organism, L.paracasei CBA L74. The World Health Organization has defined probioticsas: “Live microorganisms which when administered in adequate amountsconfer a health benefit on the host.” In some embodiments, the L.paracasei CBA L74 can be subjected to treatments that render themnon-replicating, for example, exposure to heat, dessication,y-irradiation, or uv-irradiation. A non-replicating L. paracasei CBA L74can be a dead cell or a living cell that has been rendered incapable ofcell division. A non-replicating L. paracasei CBA L74 can be an intactcell or a cell that has undergone partial or complete lysis. In someembodiments, the non-replicating cells can include a mixture of intactand lysed cells.

While we believe we understand certain events that occur uponadministration of compositions comprising or made by fermentation withL. paracasei CBA L74, the compositions of the present invention are notlimited to those that work by affecting any particular cellularmechanism. Our working hypothesis is that probiotic organisms orcompositions fermented with probiotic organisms may provide an increasedbarrier to translocation of bacteria and bacterial products acrossmucosa, competitively exclude potential pathogens, modify of hostresponse to microbial products, and enhance enteral nutrition in waysthat inhibits the growth of pathogens. The beneficial effects ofcompositions comprising non-replicating probiotic organisms may derivefor example, from metabolites produced during fermentation, for example,organic acids such as lactic acid, butyric acid or acetic acid.Alternatively or in addition, microbial DNA, e.g, unmethylated CpGdinucleotides, bacterial cell wall fragments and other sub-cellularbacterial components, such as proteins, carbohydrates and lipids, mayexert immunomodulatory effects on the mucosal immune system.

The inventors have found that isolated L. paracasei CBA L74 modulatedthe levels of both pro- and anti-inflammatory markers when assayed invitro and in vivo. Moreover, immunomodulatory effects were also observedfollowing administration of foods that had been fermented by L.paracasei CBA L74. Such immunomodulatory effects were noted even whenthe fermented foods had been treated, e.g., by heat, to render the L.paracasei CBA L74 non-replicating. Accordingly, the invention featurescompositions and methods that can be used to stimulate the intestinalimmune system. The compositions can include food products that have beenfermented by L. paracasei CBA L74. The food products can be any of awide range of foods that are amenable to fermentation by L. paracaseiCBA L74. In some embodiments, the compositions can include isolated L.paracasei CBA L74 and a physiological carrier. The carrier may be a foodproduct, but the invention is not so limiting and in some embodimentsthe carrier may be a pharmacological carrier. Also provided are methodsof making and using the compositions. The methods of the inventioninclude methods of producing compositions comprising L. paracasei CBAL74, methods of fermenting food products with L. paracasei CBA L74 andmethods of administering the compositions to generate animmunomodulatory response in a subject. The compositions may beadministered to a subject having an immature immune system, a subject atrisk for a gastrointestinal disorder or who has a gastrointestinaldisorder. The methods can be used on human subjects, for example,infants and children, or in veterinary medicine. Regardless of thesubject (whether human or non-human), any of the present methods caninclude a step of identifying the subject. For example, the methods caninclude a step of determining whether the subject is in need oftreatment.

Compositions Fermented Foods

The compositions of the invention include nutritional compositions,i.e., food products fermented by the probiotic organism, L. paracaseiCBA L74. Any food product amenable to fermentation by L. paracasei CBAL74 may be used. The food product can be a dairy product, for example,milk or a milk-based product. Exemplary milk sources include, withoutlimitation, cattle, sheep, goats, yaks, water buffalo, horses, donkeys,reindeer and camels. Regardless of the source, the milk or milk productscan be in any form suitable for fermentation by L. paracasei CBA L74.For example, the milk can be whole milk or milk that has been processedto remove some or all of the butterfat, e.g., 2% milk, 1% milk or no-fatmilk. Alternatively or in addition, the milk can be previouslypasteurized and or homogenized, dried and reconstituted, condensed orevaporated. Fractions of milk products including casein, whey protein orlactose may also be used. In some embodiments, the milk product can befrom about 1% to about 30% reconstituted skim milk powder, for exampleabout 2%, about 5%, about 7%, about 9%, about 10%, about 12%, about 15%,about 20%, about 25%, about 30% reconstituted skim milk powder. Prior tofermentation the milk product can be combined with one or more of thefollowing: a) a carbohydrate (e.g., a disaccharide such as dextrose or astarch; b) a lipid; c) a vitamin and d) a mineral. For example, skimmilk powder may be combined with dextrose to about 2%, e.g., about0.25%, about 0.50%, about 0.75%, about 1.0%, about 1.5% or about 2.0%.

The food product can be a cereal product, for example, rice, wheat,oats, barley, corn, rye, sorghum, millet, or triticale. The cerealproduct can be a whole grain or be milled into a flour. The food productcan be a single kind of cereal or a mixture of two or more kinds ofcereals, e.g., oat flour plus malted barley flour. The cereal productscan be of a grade and type suitable for human consumption or can beproducts suitable for consumption by domestic animals. Generally, thecereal product is hydrated prior to fermentation. The concentration ofcereal can vary, but useful ranges include from about 5% to about 50%weight/volume, for example, about 8% weight/volume, about 10%weight/volume, about 12% weight/volume, about 15% weight/volume, about18% weight/volume, about 20% weight/volume, about 22% weight/volume,about 25% weight/volume, about 30% weight/volume, about 35%weight/volume, about 40% weight/volume, about 45% weight/volume or about50% weight/volume. Exemplary concentrations include 15% weight/volume ofrice or a mixture of 18.5% weight/volume oat flour plus 5% weight/volumeof malted barley flour. The pH of the hydrated cereals may be adjustedusing any acid suitable for consumption. The acid can be, for example,an organic acid. Useful organic acids include acetic acid, citric acid,lactic acid, adipic acid, malic acid and tartaric acid. Any combinationof two or more acids can be used. In some embodiments, the pH may beadjusted to about 4.0 using citric acid.

The food product can also be a vegetable or a fruit product, forexample, a juice, a puree, a concentrate, a paste, a sauce, a pickle ora ketchup. Exemplary vegetables and fruits include, without limitation,squashes, e.g., zucchini, yellow squash, winter squash, pumpkin;potatoes, asparagus, broccoli, Brussels sprouts, beans, e.g., greenbeans, wax beans, lima beans, fava beans, soy beans, cabbage, carrots,cauliflower, cucumbers, kohlrabi, leeks, scallions, onions, sugar peas,English peas, peppers, turnips, rutabagas, tomatoes, apples, pears,peaches, plums, strawberries, raspberries, blackberries, blueberries,lingonberries, boysenberries, gooseberries, grapes, currants, oranges,lemons, grapefruit, bananas, mangos, kiwi fruit, and carambola.

The food product can also be a “milk” made from grains (barley, oat orspelt “milk”) tree nuts (almond, cashew, coconut, hazelnut or walnut“milk”), legumes (soy, peanut, pea or lupin “milk”) or seeds (quinoa,sesame seed or sunflower seed “milk”).

Also contemplated are food products comprising animal proteins, forexample, meat, for example, sausages, dried meats, fish and dried fishproducts.

Regardless of the type of food product that is used, the product iscombined with L. paracasei CBA L74 and incubated at a temperature andfor a time sufficient for fermentation to occur. Any standardfermentation method known in the art may be used. Specific fermentationconditions will vary according to many factors including, for example,the type of food product, the concentration of the food product, theinstrumentation that is used, the sample volume, the initialconcentration of the L. paracasei CBA L74 inoculum, the presence, ifany, of a co-inoculum, the organoleptic properties of the fermentedfood, and the intended use of the fermented food.

Both the instrumentation and the substrate (i.e., the food product to befermented) are sterilized prior to inoculation with L. paracasei CBA L74in order to decrease the level of, or eliminate, viable bacteria and/orfungi and/or infectious viruses. The instrumentation can be sterilizedusing standard methods or according to the manufacturer's instructions.Choice of a particular method for sterilization of the substrate willdepend, in part, on the stability of the substrate to the sterilizationmethod. For example, the substrate can be sterilized by steam andpressure, e.g. by autoclaving, repeated cycles of heating and cooling(e.g., tyndalization) exposure to high pressures (e.g., pascalization),ultrafiltration, or radiation (e.g., exposure to gamma-, x-, e-beam,and/or ultra-violet (wavelength of 10 nm to 320 nm, e.g., 50 nm to 320nm, 100 nm to 320 nm, 150 nm to 320 nm, 180 nm to 320 nm, or 200 nm to300 nm). Aliquots of the substrate can be removed following treatmentand plated on suitable media to confirm the absence of bacterial and/orfungal contaminants. If the substrate has been sterilized by exposure tohigh temperatures, it should be cooled to at least 37° C. prior toinoculation with L. paracasei CBA L74.

The substrate can be inoculated with L. paracasei CBA L74 according tostandard methods, for example, from fresh liquid culture or afreeze-dried culture that has been resuspended in aqueous medium for ashort time prior to inoculation. In general, L. paracasei CBA L74 areadded at concentrations of about 0.5×10⁶ to about 1×10⁶ cfu/ml ofsubstrate, e.g., about 1×10⁶ cfu/ml, about 2×10⁶ cfu/ml, about 5×10⁶cfu/ml, 7×10⁶ cfu/ml 8×10⁶ cfu/ml. The culture should be agitatedsufficiently to produce a relatively uniform distribution of bacteriaand substrate, but not excessively since L. paracasei CBA L74 is ananaerobic bacterium. For example, a five liter culture may be agitatedat about 150 rpm. Fermentation temperature is generally at 37° C.Various parameters, for example, the pH, the partial pressure of O₂,stirrer speed, temperature, gas mixing, foam level and substrateconcentration can be monitored during fermentation and adjustedaccordingly. Growth of the L. paracasei CBA L74 can be monitored usingstandard microbiological methods. Fermentation is carried out until theconcentration of L. paracasei CBA L74 is about between about 10⁸/ml andabout 10⁹/ml. Depending upon the substrate and other conditions, thisconcentration may be reached in about 10 to about 30 hours afterinoculation, e.g., about 12 hours, about 15 hours, about 18 hours, about24 hours, about 30 hours.

Samples of the substrate can be assayed before, during and afterfermentation for quality assurance using standard microbiologicalmethods. Exemplary methods include, but are not limited to, growth onRogosa agar for L. paracasei CBA L74, growth on plate count agar (PCA)for total aerobes, growth on McConkay agar for coliforms, growth onreinforced clostridial agar (RCM) for Clostridia. In addition to colonycounts, colony morphologies can be observed and compared to referencesamples.

In some embodiments, a co-inoculum can be added along with the L.paracasei CBA L74 in order to help initiate fermentation. Usefulco-inocula for fermentation of milk products include, for example,without limitation, Streptococcus thermophilus, Lactobacillus paracasei,Lactobacillus salivarious, Lactobacillus rhamnosus, Lactobacillus casei,Lactobacillus lactis, Lactobacillus delbrueckii, subsp. Bulgaricus,Lactobacillus acidophilus, Lactobacillus brevis, or Leuconostocmesenteroides. In general, the concentration of the co-inoculum will belower than that of L. paracasei CBA L74, for example, about1×10⁴/ml×10⁵/ml. The final concentration of S. thermophilus can rangefrom about 0.5×10⁸/ml to about 2.5×10⁸/ml.

Food Products

Once suitable concentrations of L. paracasei CBA L74 have been reached,the fermented food can be further processed for use. In someembodiments, the pH of the fermented food can be adjusted, for examplefrom about 3.0 to nearer to neutrality, e.g., 6.5, with the addition ofNaOH or KOH. In some embodiments the fermented food can be dried. Thefermented food product can be dried by any method known in the art thatwill result in the retention of immunomodulatory properties of thefermented food. Exemplary drying methods include spray drying,freeze-drying e.g., lyophilization, or drum-drying. The final watercontent of the fermented food product may vary but can be between about1% and about 10% or more. In some embodiments, the drying process canrender the L. paracasei CBA L74 non-replicating.

The dried fermented foods can be hydrated before use. Depending on theamount of liquid used in the hydration, the fermented food products maycontain the equivalent of about 10² and 10¹² cfu/ml of L. paracasei CBAL74. The dried L. paracasei CBA L74 do not form colonies, so it isunderstood that this amount is calculated based on the number of livebacteria that were present in the fermented foods prior to the dryingstep. In some embodiments, the fermented food products may include theequivalent of about 10⁷ to about 10¹² cfu/g, e.g., about 5×10⁷ cfu/g,about 1×10⁸ cfu/g, about 5×10⁸ cfu/g, about 1×10⁹ cfu/g, about 5×10⁹cfu/g, about 1×10¹⁰ cfu/g, about 5×10¹⁰ cfu/g, about 1×10¹¹ cfu/g, about5×10¹¹ cfu/g of dry weight.

Two or more fermented food products prepared by the methods of theinvention may be combined prior to administration. For example,fermented milk products may be combined with fermented cereal products.Alternatively, the fermented food product can be combined with otherfood products, for example, non-fermented food products or food productsfermented using other bacterial strains. Any combination can be usedprovided that the immunomodulatory properties of the fermented food areretained. Exemplary food products include, without limitation, dairyproducts, e.g., milk, yoghurt, curd, cheese and cheese-based products,fermented milks, milk-based fermented products, milk-based powders,infant formulae, milk-based strained infant foods, ice cream, gelato,puddings, soups, sauces, purees, or dressings, nutritional formulas forthe elderly; cereal products e.g., pablum, cereal-based strained infantfoods, oatmeal, farina, semolina, polenta, pasta, biscuits, crackers,energy bars; vegetable products, e.g., purees, vegetable-based strainedinfant foods, pickled vegetables including cucumbers, cabbage, carrots,beans, peppers, or relishes; fruit products, e.g., fruit-based strainedinfant foods, tomato products, purees, sauces, pastes, ketchups, fruitpurees; or a protein-based products, e.g., legumes, sausages, lunchmeats, hot dogs, or pureed meats. In some embodiments the fermented foodmay be combined with pet foods or animal feeds.

In some embodiments, the compositions can include L. paracasei CBA L74fermentates, from which all or substantially all, of the L. paracaseiCBA L74 cells have been removed. Methods for separating cells fromgrowth media are well known in the art and can rely upon physicalmethods, for example, centrifugation to produce a cell pellet and aculture supernatant, filtration, ultrafiltration, tangentialflow-filtration, normal flow filtration or reverse osmosis.Alternatively or in addition, the separation method can be ligand-basedand include, for example, an antibody that specifically binds to L.paracasei CBA L74. The antibody can be coupled to a solid support suchas a magnetic bead.

Isolated L. paracasei CBA L74

In some embodiments, the compositions of the invention include L.paracasei CBA L74 that are partially or substantially isolated from themedia in which they were grown. The L. paracasei CBA L74 can be live ornon-replicating, e.g., inactivated, for example, by heat-treatment. Thecells can be lyophilized or freeze-dried under conditions that preservecell viability. Methods of lyophilization are well known in the art.

Physiological Carriers

In some embodiments, the compositions of the invention may includeisolated L. paracasei CBA L74 in combination with a physiologicallyacceptable carrier. The L. paracasei CBA L74 can be live ornon-replicating, e.g., inactivated, for example, by heat-treatment. Thedosage may vary, but can range from the equivalent of about 10² to about10¹² cfu/g, e.g., 1×10² cfu/g, 5×10² cfu/g, 1×10³ cfu/g, 5×10³ cfu/g,1×10⁴ cfu/g, 5×10⁴ cfu/g, 1×10⁵ cfu/g, 5×10⁵ cfu/g, 1×10⁶ cfu/g, 5×10⁶cfu/g, 1×10₇ cfu/g, 5×10₇ cfu/g, 1×10⁸ cfu/g, 5×10⁸ cfu/g, 1×10⁹ cfu/g,5×10⁹ cfu/g, 1×10¹⁰ cfu/g, 5×10¹⁰ cfu/g, 1×10¹¹ cfu/g, 5×10¹¹ cfu/g,1×10¹² cfu/g of dry weight.

The physiologically acceptable carrier can be a food or food product.Isolated L. paracasei CBA L74 can be added to a food or food productprior to packaging or processing. Alternatively or in addition, isolatedL. paracasei CBA L74 can be added to a food or food product prior toconsumption. For example, isolated L. paracasei CBA L74 can be combinedwith any of the foods or food products described above. The food productcan be a fermented food product or an unfermented food product. Forexample, isolated L. paracasei CBA L74 can be added to an unfermenteddairy or cereal product. In some embodiments, the L. paracasei CBA L74can be added to a food or food product to include the equivalent ofabout 10⁷ to about 10¹² cfu/g, e.g., about 5×10⁷ cfu/g, about 1×10⁸cfu/g, about 5×10⁸ cfu/g, about 1×10⁹ cfu/g, about 5×10⁹ cfu/g, about1×10¹⁰ cfu/g, about 5×10¹⁰ cfu/g, about 1×10¹¹ cfu/g, about 5×10¹¹ cfu/gof dry weight.

Pharmaceutical Carriers

The compositions also include a pharmaceutically acceptable carrier. Weuse the terms “pharmaceutically acceptable” (or “pharmacologicallyacceptable”) to refer to molecular entities and compositions that do notproduce an adverse, allergic or other untoward reaction whenadministered to an animal or a human, as appropriate. The term“pharmaceutically acceptable carrier,” as used herein, includes any andall solvents, dispersion media, coatings, antibacterial, isotonic andabsorption delaying agents, buffers, excipients, binders, lubricants,gels, surfactants and the like, that may be used as media for apharmaceutically acceptable substance.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, the L. paracasei CBA L74 described herein, incombination with one or more pharmaceutically acceptable carriers. Insome embodiments, the L. paracasei CBA L74 can be sterilized usingconventional sterilization techniques before or after it is combinedwith the pharmaceutically acceptable carrier. In making the compositionsof the invention, the L. paracasei CBA L74 is typically mixed with anexcipient, diluted by an excipient or enclosed within such a carrier inthe form of, for example, a capsule, tablet, sachet, paper, or othercontainer. When the excipient serves as a diluent, it can be a solid,semisolid, or liquid material (e.g., normal saline), which acts as avehicle, carrier or medium for the active ingredient. Thus, thecompositions can be in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosols (as a solid or in a liquid medium), ointments, soft and hardgelatin capsules, suppositories, sterile injectable solutions, andsterile packaged powders. As is known in the art, the type of diluentcan vary depending upon the intended route of administration. Theresulting compositions can include additional agents, such aspreservatives. The excipient or carrier is selected on the basis of themode and route of administration. Suitable pharmaceutical carriers, aswell as pharmaceutical necessities for use in pharmaceuticalformulations, are described in Remington's Pharmaceutical Sciences (E.W. Martin), a well-known reference text in this field, and in the USP/NF(United States Pharmacopeia and the National Formulary). Some examplesof suitable excipients include lactose, dextrose, sucrose, sorbitol,mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thepharmaceutical compositions can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

Pharmaceutically acceptable compositions for use in the present methods,including those in which L. paracasei CBA L74 is entrapped in a colloidfor oral delivery, can be prepared according to standard techniques. TheL. paracasei CBA L74 can be dried and compacted by grinding orpulverizing and inserted into a capsule for oral administration. In someembodiments, the L. paracasei CBA L74 can be combined one or moreexcipients, for example, a disintegrant, a filler, a glidant, or apreservative. Suitable capsules include both hard shell capsules orsoft-shelled capsules. Any lipidbased or polymer-based colloid may beused to form the capusule. Exemplary polymers useful for colloidpreparations include gelatin, plant polysaccharides or their derivativessuch as carrageenans and modified forms of starch and cellulose, e.g.,hypromellose. Optionally, other ingredients may be added to the gellingagent solution, for example plasticizers such as glycerin and/orsorbitol to decrease the capsule's hardness, coloring agents,preservatives, disintegrants, lubricants and surface treatment. In someembodiments, the capsule does not include gelatin. In other embodiments,the capsule does not include plant polysaccharides or their derivatives.

Regardless of their original source or the manner in which they areobtained, the L. paracasei CBA L74 of the invention can be formulated inaccordance with their use. These compositions can be prepared in amanner well known in the pharmaceutical art, and can be administered bya variety of routes, depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may be oralor topical (including ophthalmic and to mucous membranes includingintranasal, vaginal and rectal delivery). In some embodiments,administration can be pulmonary (e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal) or ocular. Methods for ocular delivery caninclude topical administration (eye drops), subconjunctival, periocularor intravitreal injection or introduction by balloon catheter orophthalmic inserts surgically placed in the conjunctival sac. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular administration. Parenteraladministration can be in the form of a single bolus dose, or may be, forexample, by a continuous perfusion pump. Pharmaceutical compositions andformulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquids,powders, and the like. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable.

The compositions can be formulated in a unit dosage form, each dosagecontaining, for example, from about 0.005 mg to about 2000 mg of L.paracasei CBA L74 per daily dose. The term “unit dosage forms” refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient. For preparingsolid compositions such as tablets, the principal active ingredient ismixed with a pharmaceutical excipient to form a solid preformulationcomposition containing a homogeneous mixture of a compound of thepresent invention. When referring to these preformulation compositionsas homogeneous, the active ingredient is typically dispersed evenlythroughout the composition so that the composition can be readilysubdivided into equally effective unit dosage forms such as tablets,pills and capsules. This solid preformulation is then subdivided intounit dosage forms of the type described above containing from, forexample, 0.005 mg to about 1000 mg of the L. paracasei CBA L74 of thepresent invention.

The compositions can be formulated in a unit dosage form, each dosagecontaining, for example, from about 0.1 mg to about 50 mg, from about0.1 mg to about 40 mg, from about 0.1 mg to about 20 mg, from about 0.1mg to about 10 mg, from about 0.2 mg to about 20 mg, from about 0.3 mgto about 15 mg, from about 0.4 mg to about 10 mg, from about 0.5 mg toabout 1 mg; from about 0.5 mg to about 100 mg, from about 0.5 mg toabout 50 mg, from about 0.5 mg to about 30 mg, from about 0.5 mg toabout 20 mg, from about 0.5 mg to about 10 mg, from about 0.5 mg toabout 5 mg; from about 1 mg from to about 50 mg, from about 1 mg toabout 30 mg, from about 1 mg to about 20 mg, from about 1 mg to about 10mg, from about 1 mg to about 5 mg; from about 5 mg to about 50 mg, fromabout 5 mg to about 20 mg, from about 5 mg to about 10 mg; from about 10mg to about 100 mg, from about 20 mg to about 200 mg, from about 30 mgto about 150 mg, from about 40 mg to about 100 mg, from about 50 mg toabout 100 mg of the active ingredient.

In some embodiments, tablets or pills of the present invention can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer which serves to resist disintegration inthe stomach and permit the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol, and cellulose acetate.

The liquid forms in which the compositions of the present invention canbe incorporated for administration orally or by injection includeaqueous solutions, suitably flavored syrups, aqueous or oil suspensions,and flavored emulsions with edible oils such as cottonseed oil, sesameoil, coconut oil, or peanut oil, as well as elixirs and similarpharmaceutical vehicles.

The proportion or concentration of the compositions of the invention ina pharmaceutical composition can vary depending upon a number of factorsincluding dosage, chemical characteristics (e.g., hydrophobicity), andthe route of administration. For example, the L. paracasei CBA L74 ofthe invention can be provided in a capsule containing from about 0.005mg gram to about 1000 mg for oral administration. Alternatively or inaddition, the dosage can be expressed as cfu/g of dry weight. The dosagemay vary, but can range from the equivalent of about 10² to about 10¹²cfu/g, e.g., 1×10² cfu/g, 5×10² cfu/g, 1×10³ cfu/g, 5×10³ cfu/g, 1×10⁴cfu/g, 5×10⁴ cfu/g, 1×10⁵ cfu/g, 5×10⁵ cfu/g, 1×10⁶ cfu/g, 5×10⁶ cfu/g,1×10⁷ cfu/g, 5×10⁷ cfu/g, 1×10⁸ cfu/g, 5×10⁸ cfu/g, 1×10⁹ cfu/g, 5×10⁹cfu/g, 1×10¹⁰ cfu/g, 5×10¹⁰ cfu/g, 1×10¹¹ cfu/g, 5×10¹¹ cfu/g, 1×10¹²cfu/g of dry weight.

Methods of Use

The compositions disclosed herein are generally and variously useful forstimulation of an immunomodulatory response in the mucosal immunesystem. Subjects for whom such stimulation is beneficial include thosehave a mucosal immune system deficit, for example, those having animmature immune system, such as infants or small children, those havinga depressed immune system, such as the elderly, patients takingimmunosuppressive drugs, radiation or chemotherapy, those having ahyperactivated immune system due to allergies or autoimmune disordersand those suffering from gastrointestinal disorders. Gastrointestinaldisorders can include infections due to viruses, e.g., rotaviruses;pathogenic bacteria, e.g., Salmonella, Yersinia, Shigella, Listeria,Clostridium, E. coli, E. sakazaki, H. pylori; or pathogenic protozoa,e.g., Entamoeba histolytica, Cryptosporidium spp, Campylobacter spp.Gastrointestinal disorders can also include, for example, foodallergies, food hypersensitivity, irritable bowel syndrome, inflammatorybowel disease, pouchitis, Crohn's disease, ulcerative colitis, celiacdisease, necrotizing enterocolitis, and aging, particularly aging of thegastrointestinal system,

A subject is effectively treated whenever a clinically beneficial resultensues. This may mean, for example, a complete resolution of thesymptoms associated with a mucosal immune system deficit, a decrease inthe severity of the symptoms associated with a mucosal immune systemdeficit, or a slowing of the progression of symptoms associated with amucosal immune system deficit. These methods can further include thesteps of a) identifying a subject (e.g., a patient and, morespecifically, a human patient) who has a mucosal immune system deficit;and b) providing to the subject a composition comprising L. paracaseiCBA L74 described herein, such as any fermented food product orcomposition comprising L. paracasei CBA L74 in a physiologicallyacceptable carrier. An amount of such a composition provided to thesubject that results in a complete resolution of the symptoms associatedwith a mucosal immune system deficit, a decrease in the severity of thesymptoms associated with a mucosal immune system deficit, or a slowingof the progression of symptoms associated with a mucosal immune systemdeficit is considered a therapeutically effective amount. The presentmethods may also include a monitoring step to help optimize dosing andscheduling as well as predict outcome.

The methods disclosed herein can be applied to a wide range of species,e.g., humans, non-human primates (e.g., monkeys), horses, pigs, cows orother livestock, dogs, cats or other mammals kept as pets, rats, mice,or other laboratory animals. The compositions described herein areuseful in therapeutic compositions and regimens or for the manufactureof a medicament for use in treatment of conditions as described herein(e.g., a mucosal immune system deficit due to immaturity, aging,infection, food allergies, an inflammatory or autoimmune disorder.)

The nutritional compositions described herein can be administered orallyas part of the ordinary daily diet of a subject. The food compositionsmay be administered as nutritional support to both children and adults.When formulated as pharmaceuticals, the compositions can be administeredto any part of the host's body for subsequent delivery to a target cell.A composition can be delivered to, without limitation, the brain, thecerebrospinal fluid, joints, nasal mucosa, blood, lungs, intestines,muscle tissues, skin, or the peritoneal cavity of a mammal. In terms ofroutes of delivery, a composition can be administered by intravenous,intracranial, intraperitoneal, intramuscular, subcutaneous,intramuscular, intrarectal, intravaginal, intrathecal, intratracheal,intradermal, or transdermal injection, by oral or nasal administration,or by gradual perfusion over time. In a further example, an aerosolpreparation of a composition can be given to a host by inhalation.

Regardless of whether the compositions are formulated as food productsor as pharmaceuticals, the dosage required will depend on the route ofadministration, the nature of the formulation, the nature of thesubject's condition, e.g., immaturity of the immune system or agastrointestinal disorder, the subject's size, weight, surface area,age, and sex, other drugs being administered, and the judgment of theattending clinicians. Suitable dosages are in the range of 0.01-1.000mg/kg. Some typical dose ranges are from about 1 μg/kg to about 1 g/kgof body weight per day. In some embodiments, the dose range is fromabout 0.01 mg/kg to about 100 mg/kg of body weight per day. In someembodiments, the dose can be, for example, 1 mg/kg, 2 mg/kg, 5 mg/kg, 10mg/kg, 20 mg/kg, 50 mg/kg or 100 mg/kg. The dosage is likely to dependon such variables as the type and extent of progression of the diseaseor disorder, the overall health status of the particular patient, therelative biological efficacy of the compound selected, formulation ofthe excipient, and its route of administration.

Effective doses can be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems. For example, in vitroanalysis of cytokine production by peripheral blood mononuclear cells(PBMCs) can be a useful for assaying pro- and anti-inflammatoryresponses, e.g., secretion of IL-1β, IL-12, IL-4, TNF-α, or IL-10respectively. Compositions can also be analyzed for effects in animalmodels, for example, IgA production, cytokine production by explants ofPeyer's patches, and dendritic cell and T-cell responses.

Wide variations in the needed dosage are to be expected in view of thevariety of cellular targets and the differing efficiencies of variousroutes of administration. Variations in these dosage levels can beadjusted using standard empirical routines for optimization, as is wellunderstood in the art. Administrations can be single or multiple (e.g.,2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold).Encapsulation of the compounds in a suitable delivery vehicle (e.g.,polymeric microparticles or implantable devices) may increase theefficiency of delivery.

The duration of treatment with any composition provided herein can beany length of time from as short as one day to as long as the life spanof the host (e.g., many years). For example, a composition can beadministered once a week (for, for example, 4 weeks to many months oryears); once a month (for example, three to twelve months or for manyyears); or once a year for a period of 5 years, ten years, or longer. Itis also noted that the frequency of treatment can be variable. Forexample, the present compositions can be administered once (or twice,three times, etc.) daily, weekly, monthly, or yearly. When thecompositions are formulated as food product, for example, thecompositions can be administered daily at every meal.

Any method known to those in the art can be used to determine if aparticular response is induced. Clinical methods that can assess thedegree of a particular disease state can be used to determine if aresponse is induced. For example, a subject can be monitored forsymptomatic relief, e.g., relief from colic, diarrhea, constipation,nausea, vomiting, abdominal pain, cramping, heartburn, abdominaldistention, flatulence, or incontinence. Alternatively or in addition,serum markers, imaging techniques, e.g., ultrasound, x-rays, andendoscopic methods can be used.

The compositions may also be administered in conjunction with othertherapeutic modalities. Other therapeutic modalities will vary accordingto the particular disorder, but can include, for example, anti-diarrheamedications, anti-emetics, anti-cholinergic agents, anti-inflammatoryagents, antibiotics anti-histamines and other dietary treatments, forexample, hypoallergenic infant formulas. Concurrent administration oftwo or more therapeutic agents does not require that the agents beadministered at the same time or by the same route, as long as there isan overlap in the time period during which the agents are exerting theirtherapeutic effect. Simultaneous or sequential administration iscontemplated, as is administration on different days or weeks.

Articles of Manufacture

The compositions described herein can also be assembled in kits,together with instructions for use. Accordingly, packaged products(e.g., containers containing one or more of the L. paracasei CBA L74compositions described herein and packaged for storage, shipment, orsale at concentrated or ready-to-use concentrations) and kits, includingat least one compound of the invention and instructions for use, arealso within the scope of the invention. In any of the packaged productsor kits, the L. paracasei CBA L74 compositions can include L. paracaseiCBA L74 that have been rendered non-replicating. For example, the kitscan include measured amounts of a nutritional composition including oneor more food products fermented with L. paracasei CBA L74. Theinstructions for use can be conveyed by any suitable media. For example,they can be printed on a paper insert in one or more languages orsupplied audibly or visually (e.g., on a compact disc). The packagingmaterials can include packaging materials, for example, vials, packets,containers. In some embodiments, the kits can include measured amountsof a composition comprising L. paracasei CBA L74 in a physiologicallyacceptable carrier along with packaging materials and instructions foruse in any of the formats described above. In some embodiments, the kitscan include containers containing one or more L. paracasei CBA L74compositions, e.g., L. paracasei CBA L74 and a pharmaceutical carrier,and one or more of a suitable stabilizer, carrier molecule, flavoring,and/or the like, as appropriate for the intended use. A product caninclude a container (e.g., a vial, jar, bottle, bag, or the like)containing one or more L. paracasei CBA L74 compositions. In addition,an article of manufacture further may include, for example, packagingmaterials, instructions for use, syringes, buffers or other controlreagents for treating or monitoring the condition for which prophylaxisor treatment is required. The product may also include a legend (e.g., aprinted label or insert or other medium describing the product's use(e.g., an audio- or videotape)). The legend can be associated with thecontainer (e.g., affixed to the container) and can describe the mannerin which the compound therein should be administered (e.g., thefrequency and route of administration), indications therefor, and otheruses. The components of the kit may be suitable for immediate use. Thecompounds can be ready for administration (e.g., present indose-appropriate units), and may include a pharmaceutically acceptableadjuvant, carrier or other diluent and/or an additional therapeuticagent. The invention encompasses kits, however, that includeconcentrated formulations and/or materials that may require dilutionprior to use. Alternatively, the compounds can be provided in aconcentrated form with a diluent and instructions for dilution. Thecomponents of the kit may be suitable for immediate use. The inventionencompasses kits, however, that include concentrated formulations and/ormaterials that may require dilution prior to use.

EXAMPLES Example 1: Isolation and Characterization of L. paracasei CBAL74

We analyzed different strains of Lactobacilli for their ability toferment aqueous suspensions containing different concentrations of riceflour or wheat flour. L. paracasei CBA L74 was selected for furtheranalysis based on the low pH values and high CFU counts. This strain wasdeposited under the Budapest Treaty on the International Recognition ofthe Deposit of Micro-organisms for the Purposes of Patent Procedure onSep. 9, 2008 at the Belgian Coordinated Collections of Microorganisms(BCCM) Laboratorium voor Microbiologie (LMG), Ghent, Belgium. TheAccession Number given by the International Depositary Authority is LMGP-24778.

Example 2: Preparation of L. paracasei CBA L74 Fermented Milk

Conditions:

-   -   Substrate: 9% reconstituted skim milk powder, dextrose added at        0.25%    -   Substrate heat treatment: UHT—135° C. for 3 s or equivalent F₀    -   Co-Inoculum: 5×10⁶ for Lactobacillus paracasei CBA-L74        -   5×10⁴ for Streptococcus thermophilus (as starter of the            fermentation)    -   Fermentation Temperature: 37° C.    -   Fermentation time: 15 h hours    -   pH during fermentation: no adjustment    -   At the end of the fermentation pH adjustment to 6.5 with NaOH        solution    -   Spray drying with inlet temperature 190° C. and outlet        temperature 90° C.    -   Analysis: Cells count on the fermentate to determine        Streptococcus thermophiles and Lactobacillus paracasei CBA-L74

Plating:

Lactobacilli selective agar (LBS) was used for detection ofLactobacillus paracasei CBA-L74. L-M17 agar was used for Streptococcusthermophilus counts. Both were incubated at 37° C. anaerobically. Platecount agar (PCA) was used for detection of contaminants and incubated at30° C. aerobically.

Fermentation:

L. paracasei CBA L74 and S. thermophilus 1773 co-inoculum were added asfresh cultures. Fermentation was carried out for 15 hours, to aconcentration of 10⁸ cfu/ml of L. paracasei CBA-L74. The initial pH was6.6. At the end of the fermentation the pH was 5.1. The pH was adjustedto 6.5 by adding 2.5N NaOH. The initial concentration of L. paraceseiCB-74 was 5×10⁶ CFU/ml; the final concentration was more than 10⁸CFU/ml. The initial concentration of Streptococcus thermophilus was5×10⁴ CFU/ml; the final concentration was 1×10⁸ CFU/ml. The initialtotal bacterial count on PCA was 0 in the milk prior to inoculation andat T0 and too few colonies to count (TFTC) after the 15 hourfermentation period. was 5×10⁴ CFU/ml, the final concentration was 1×10⁸CFU/ml.

Drying:

The fermentate was dried at an inlet temperature of 190° C. and anoutlet temperature of 90° C. The moisture content of the powder afterspray drying was 4.87%.

Example 3: Preparation of L. paracasei CBA L74 Fermented Oat and BarleyFlour

We prepared a one liter solution of 18.5% (w/vol) oat flour+5% (w/w)malted barley flour, using 185 g oat flour and 9.25 g malted barleyflour. The mixture of flour and water was adjusted to pH 4.00 with 0.5 Mcitric acid. The fermenter was sterilized by autoclaving. Then themixture of flour+ water+ citric acid was added to the fermenter.

The mixture was heat-treated at 80° C. for 30 minutes then cooled downto 37° C. Three different sets of fermentations conditions were tested.All fermentations were terminated after 16 hours, a time that coincidedwith the end of log phase growth.

Trial #1:

L. paracasei CBA L74 was added to the heat-treated cereal solution to afinal concentration of 2.3×10⁶ CFU/ml and incubated with agitation at37° C. After 16 hours count plate in MRS was 7.6×10⁸ UFC/ml (lactic acidbacteria); contaminants measured in PCA, MC and SB were below 1000UFC/ml. The final pH was 3.8. After 20 hours count plate in MRS was1.2×10⁸ UFC/ml (lactic acid bacteria); contaminants were absent. After24 hours count plate in MRS was 5×10⁸ UFC/ml (lactic acid bacteria);contaminants were absent. Because log phase stop after 16 hours, it waspreferable to stop fermentations at 16 hours.

Trial #2 (pH Stabilized)

This fermentation was carried out by keeping the pH at 4 using 2N NaOHL. paracasei CBA L74 was added to the heat-treated cereal solution to afinal concentration of 2.1×10⁶ CFU/ml and incubated with agitation at37° C. After 16 hours count plate in MRS was 7.5×10⁸ UFC/ml (lactic acidbacteria); contaminants measured in PCA, MC and SB were below 1000UFC/ml.

Trial #3 (pH Stabilized)

This fermentation was carried out by keeping the pH at 4 using 2N NaOHL. paracasei CBA L74 was added to the heat-treated cereal solution to afinal concentration of 5.1×10⁶ CFU/ml and incubated with agitation at37° C. After 16 hours count plate in MRS was 2.1×10⁹ UFC/ml (lactic acidbacteria); contaminants measured in PCA, MC and SB were below 1000UFC/ml.

Example 4: Preparation of L. paracasei CBA L74 Fermented Rice and WheatFlour

We prepared a one liter solution of 15% weight/volume of rice bycombining 150 g of rice and 900 ml of water. The mixture was prepared atroom temperature and mixed by shaking for several minutes at 1000-1300rpm. The rice mixture was treated by tyndalization by heating of themixture inside the instrument at 70° C., starvation at 70° C. per 20-30minutes, cooling at 30-37° C., starvation at 30-37° C. per 20-30minutes, heating at 70° C., starvation at 70° C. per 20-30 minutes,cooling at the fermentation temperature (37° C.) while shaking at150-600 rpm.

L. paracasei CBA L74 was added from a freeze-dried sample to a finalconcentration of 1×10⁶ CFU/ml. The freeze-dried sample was resuspendedin water and incubated briefly at 37° C. to activate the bacteria. Afterthe inoculation, the mixture was homogenized by shaking briefly at300-600 rpm; during fermentation the solution was shaken at 150 rpm.Fermentation was carried out at 37° C. for 24 hours at a pO2 of <15%.Aliquots were collected at the time of inoculation (T0), at 16 hours(T16), 18 hours (T18), 21 hours (T21) and at 24 hours (T24). Afterfermentation, the cereal was heated to 50° C. with continuous mixing.The heated cereal was then spray dried at T air_(in) 80° C., T air_(out)210° C. The final moisture content was 6%.

Samples were analyzed on Rogosa agar(+vancomycin 12 microgr./ml) (48 hat 37° C.), for quantification of the L. paracasei CBA L74), on PCA fortotal aerobes (24 h at 37° C.), on McConkay agar for coliformes and RCMagar for clostridia.

The results of this fermentation were as follows:

inoculum (L. paracasei CBA L74): 1×10₆(+/−½ log) CFU/ml (on theinstrument)L. paracasei CBA L74 concentration after 24 hours of fermentation:1×10⁸(+/−½ log) CFU/mlContaminants on PCA before inoculum: <10⁴ CFU/mlContaminants on McConkay before inoculum: <10⁴ CFU/mlContaminants on RCM before inoculum: <10 CFU/mlContaminants on PCA after inoculum: <10⁴ CFU/mlContaminants on PCA after 24 hours of fermentation: <10⁴ CFU/mlpH before the addition of inoculum: 6 (+/−0.20)pH at 16-18 hours: 3.70 (+/−0.20)pH at 24 hours: 3.60 (+/−0.20).

Example 5: Effects of L. paracasei CBA L74 on Dendritic Cells in a Caco2Cell Coculture System

We analyzed the effect of live and non-replicating L. paracasei CBA L74in a co-culture of intestinal epithelial cells (Caco2 cells) and humandendritic cells (DCs). Caco2 cells were seeded on a transwell membraneand after about 3 weeks, when the trans-epithelial resistance wasadequate, were supplemented with L. paracasei CBA L74 for 96 hours.Human DCs were differentiated from peripheral blood monocytes and seededin the basal compartment of the co-culture chamber. The trans-epithelialresistance remained constant during the experiment.

To characterize DCs phenotype, we monitored DC cell surface expressionof co-stimulatory molecules (CD80 and CD86), MHC-II and adhesionmolecules (CD40) by cytofluorimetric analysis. To determine the cytokineprofile produced by DCs cocultured with intestinal epithelial cellsexposed to L. paracasei CBA L74, we collected the culture medium andquantified IL-10 and IL-12p70 by ELISA. To verify the ability of Caco2cells exposed to L. paracasei CBA L74 to condition DCs to promote theproliferation of T cells we performed FACS analysis and mixed lymphocytecultures.

As shown in the Table in FIG. 1 incubation of Caco2 cells for 24 hrswith L. paracasei CBA L74 alive or inactivated (thermal inactivation)modified the phenotype of co-cultured dendritic cells. Furthermore thepresence of L. paracasei CBA L74 modulated LPS-mediated changes in DCphenotype.

We then quantified the cytokines released from DC co-cultured with Caco2cells conditioned with L. paracasei (alive or inactivated). As shown inFIG. 2A, DCs co-cultured with Caco2 exposed to L. paracasei CBA L74alive or inactivated showed statistically significant increase in IL-10production. We did not observe a significant increase in IL-12production in the absence of LPS (FIG. 2B). However, the DCs retainedthe ability to respond to LPS challenge by enhanced IL-12 production,suggesting that exposure to L. paracasei CBA L74 did not affect theoverall ability of DCs to respond to pathogens.

We then conducted a functional assay to verify the ability of DC exposedto CaCo2 cells cultured in presence of medium or medium supplementedwith L. paracasei to modulate the ability of T cells to proliferatefollowing a mixed lymphocyte reaction. As shown in FIG. 3, we did notobserve significant differences in the ability of DC co-cultured withCaCo2 cells to modify T-cells proliferation.

Taken together, the in vitro data indicate that L. paracasei CBA L74,alive or heat inactivated, can influence the environment generated byintestinal epithelial cells that in turn modulates the activity of otherimmune cells such as DC. The overall picture indicates that DCs exposedto Caco2-conditioned medium reduces the expression of activationmarkers, produce anti-inflammatory cytokines as IL-10 while retainingthe ability to respond to LPS by enhancing IL-12 production.

Example 6: Effects of L. paracasei CBA L74 on Morphology, CytokineExpression and Innate Immunity in Whole Intestinal Mucosa

We examined the in vivo effects by administering L. paracasei CBA L74(live and heat-inactivated) as a dietary supplement in mice. After twoweeks of supplementation, the animals were sacrificed and the wholeintestinal mucosa was analyzed.

Mucosal Morphology:

We performed a haematoxylin and eosin staining of paraffin embeddedileal sections. None of the supplements had significant effects onintestinal architecture or caused an infiltrate of inflammatory cells.

Cytokine and IgA Expression:

We then determined whether administration of L. paracasei CBA L74 (aliveor inactivated) affected the level of anti- and proinflammatorycytokines in the intestinal mucosa. As shown in FIG. 4, theadministration of L. paracasei CBA L74 alive or inactivatedsignificantly decreased the level of IL-1β, a potent pro-inflammatorymediator, in the intestinal mucosa of mice. As shown in FIG. 5, we alsoobserved a reduction in basal mucosal IL-4 level followingadministration of L. paracasei CBA L74 alive or inactivated. Finally, wedetermined the effect of different regimen supplementation on mucosalIgA level. Following two weeks of diet supplementation with L. paracaseiCBA L74, animals were sacrificed and the intestinal mucosa collected andhomogenized. Total IgA was then measured by ELISA and values normalizedto total mucosal proteins. As shown in FIG. 6, heat killed L. paracaseiCBA L74 significantly increased mucosal IgA.

Innate immunity: We analyzed the effect of dietary supplementation onlevels of Toll-Like Receptor 2, 4 and 9. These receptors are involved inthe recognition of conserved bacterial structures and thus play a keyrole in modulating the reactivity of immune and non-immune cells towardmicrobial conserved structures. As shown in FIG. 7, diet supplementationwith L. paracasei CBA L74, live or heat inactivated, increased levels ofprotein expression of TLR2 and TLR4. We also assayed the effect ofdietary supplementation on levels of PPARγ in the mucosa. As shown inFIG. 8, Live L. paracasei CBA L74 significantly increased the level ofPPARγ.

Example 7: Effects of L. paracasei CBA L74 on Levels of CirculatingCytokines

To assess whether the diet supplements were able to modify the level ofcirculating cytokines, we measured the effects of the dietaryadministration of the strain (alive or inactivated) on anti- andpro-inflammatory cytokines level in the serum. As shown in FIGS. 9A and9B, Live L. paracasei CBA L74 induced a statistically significantdecrease in IL-1β and a modest increase in circulating IL-4,respectively.

Example 8: Effects of L. paracasei CBA L74 on Dendritic Cell andT-Lymphocyte Activity

We next focused on the impact of dietary supplementation with L.paracasei CBA L74 on immune cells relevant to the activity ofmucosal-associated immune system, namely dendritic cells andlymphocytes. We evaluated the phenotype of DCs within the Peyers Patches(PP), since these cells are instrumental in establishing the fate of anantigen and contribute to the environment that will determine the natureof the adaptive immune response.

As shown in the Table in FIG. 10, L. paracasei CBA L74 supplementation(live or inactivated) decreased the expression of the co-stimulatorymolecule CD80 and of the adhesion molecule CD40 whereas MHCII expressionwas up-regulated. These data suggested that the DCs from L. paracaseiCBA L74 supplemented animals seemed less ready to interact with T-cellsand to mount an immune response but their ability to process and presentantigens was preserved.

We then determined the ability of diet supplementation with L. paracaseiCBA L74 to modify the reactivity of DCs to pro-inflammatory stimuli(such as bacterial LPS and CpG). Exposure of DCs from control mice toLPS or CpG induced a strong up-regulation of CD80 in control DCs.Supplementation with heat inactivated L. paracasei CBA L74 DC did notmodify the reactivity to inflammatory stimuli. Supplementation with livebacteria significantly reduced LPS- and CpG-induced CD80 up-regulation.

Finally, we investigated whether dietary supplementation with L.paracasei CBA L74 (live or inactivated) affected intestinalT-lymphocytes (either CD4+ and CD8+) polarization toward a Th1 or Th2phenotype. For these studies, Peyer's Patches were exposed to PHA, astrong, non-specific stimulus and then lymphocyte polarization wasevaluated by intrakine staining for IL-4 and IFN-γ. As shown in FIG.11A, in the absence of PHA (the “basal condition”) in CD4+ lymphocytes,IL-4 and IFN were almost in equilibrium. About 10-12% of the cells werepositive for these cytokines. As shown in FIG. 11B, for CD8+ lymphocytesin the basal condition, there was a slight predominance of IL-4 over IFNexpressing cells. Exposure of either CD4+ or CD8+ lymphocytes to PHAcaused a strong increase in intracellular staining for IL-4 and IFN-γwith bias toward IFN-γ production.

In CD4+ cells, dietary supplementation with live L. paracasei CBA L74increased IFN-γ levels in the basal condition. Following PHA exposurethere were no significant differences in the response among thesupplemented groups (FIG. 11A). In CD8+ lymphocytes, oralsupplementation with inactivated L. paracasei CBA L74 favored a Th2profile with a stimulation of IL-4 production over IFN-γ production(FIG. 11B) The anti-inflammatory profile is further supported by theblunted response to PHA. A similar trend was evident also in CD8+lymphocytes isolated from mice supplemented with live L. paracaseiCBA-L74, although less pronounced.

Example 9: Effect of Milk Fermented by L. paracasei CBA L74 on ImmuneSystem Markers in the Intestinal Mucosa

Mice were supplemented twice a day for two weeks with either: 1) Control(PBS); 2) Skim milk (not fermented); 3) Skim milk fermented with L.paracasei CBA L74 (1×10⁸ cfu/ml), 4) Skim milk fermented with S.thermophilus (6.7×10⁶ cfu/ml); 5) Skim milk fermented with L. paracaseiCBA L74 (1×10⁸ cfu/ml) and S. thermophilus (1.18×10⁷ cfu/ml), 6) Skimmilk fermented with L. paracasei CBA L74 (1.9×10⁹ cfu/die) and S.thermophilus (2.2×10⁸ cfu/ml). At the end of the treatment animals weresacrificed and intestinal mucosa and Peyer's Patches were collected andprocessed for analysis as described below.

Levels of Cytokines in the Intestinal Mucosa.

As shown in FIG. 12, supplementation with skim milk fermented with L.paracasei CBA L74 caused a significant increase IL-10 levels in theintestinal mucosa. Administration of skim milk fermented with S.thermophilus induced a slight reduction as compared to controls.Administration of milk fermented with both strains at higher dosageinduced a 2.1-fold increase in mucosal IL-10. None of the treatments hadany significant effect on mucosal IL-1β although supplementation withskim milk fermented with L. paracasei CBA L74 caused a slight increasein mucosal levels of this cytokine.

Myeloperoxidase Activity in the Intestinal Mucosa.

Myeloperoxidase (MPO) activity was assayed in intestinal mucosahomogenates. Administration of non fermented or fermented milk withdifferent bacteria did not induce statistically significant differencesin the myeloperoxidase activity, although there was a slight increase inMPO activity observed in the mucosa of animals receiving non fermentedmilk.

Levels of IgA in the intestinal mucosa.

As shown in FIG. 14, supplementation with non-fermented skim milk causeda significant increase in the intestinal mucosa IgA. This increase wasnot observed in animals supplemented with skim milk fermented witheither L. paracasei CBA L74 or S. thermophilus alone. Administration ofmilk fermented with both strains at higher dosage induced an increase inmucosal IgA.

Levels of TLR2, TLR4, TLR9 and PPARγ in the Intestinal Mucosa.

We next determined by WB the mucosal levels of key receptors of innateimmune system. As shown in FIGS. 15A and 15B, none of the treatments hadany significant effect on TLR4 receptor expression. Non-fermented milkcaused a modest increase in TLR2. Administration of skim milk fermentedwith either L. paracasei CBA L74 or S. thermophilus alone or incombination at the lower dose prevented this effect. A modest increase,comparable to milk alone, in TLR2 was also evident in mice receivingmilk fermented with both strains at higher dosage. Administration ofskim milk alone had profound effects on TLR9 levels, whereasadministration of fermented milk with both strains re-establishedmucosal levels comparable to controls. As shown in FIGS. 15A and 15B,administration of non-fermented milk per se caused a strong increase inmucosal PPARγ. A similar effect was evident in mice supplemented withskim milk fermented with L. paracasei CBA L74 and the lower dose of milkfermented with both strains.

Levels of pNF-kB and IKB in the Intestinal Mucosa.

We determined the activation status of NF-κB, a key transcription factorinvolved in both intestinal inflammation and epithelial cell survival.As shown in FIG. 16, activated NF-κB (pNF-κB) was detectable in thecontrol, normal mucosa. Following the different treatments, we observedonly a slight increase in pNF-κB levels in the mucosa of mice treatedwith skim milk fermented with either L. paracasei CBA L74 or S.thermophilus alone, whereas in mice receiving skim milk fermented withboth strains, pNF-κB was comparable to controls. The increase in pNF-κBparalleled the disappearance of the inhibitory subunit of NF-κB, IKB,that was not detectable in the mucosa of mice supplemented with skimmilk fermented with either L. paracasei CBA L74 or S. thermophilusalone. Mice receiving skim milk fermented with both strains showed IkBlevels comparable to controls.

Example 10: Effect of Milk Fermented by L paracasei CBA L74 on DendriticCell Phenotype in the Intestinal Mucosa

Phenotype of Dendritic Cells Extracted from Peyer's Patches.

We examined the effect of diet supplementation on the phenotype ofintestinal DCs. The results of this analysis are shown in FIG. 17.Administration of milk fermented with L. paracasei alone as well as withL. paracasei and S. thermophilus at lower and higher doses produced astatistically significant decrease in CD80 and CD86 expression ascompared to control mice (* means p<0.05 vs control), whereas HLAII andCD40 levels overall remained stable. Data in FIG. 17 are expressed asmean±S.E. of three separate experiments.

Responses of Dendritic Cells Exposed to LPS/CpG.

We determined the ability of diet supplementation to modify thereactivity of DCs to pro-inflammatory stimuli (such as bacterial LPS andCpG). As shown in FIG. 18, exposure of isolated DCs isolated fromcontrol animals to LPS and CpG caused a significant increase in theexpression of CD80 (* means p<0.05 vs un-stimulated DCs from controlmice). A similar effect was observed in DCs purified from mice receivingnon-fermented milk (° means p<0.05 vs un-stimulated DCs from micetreated with non-fermented milk). Administration of skim milk fermentedwith L. paracasei CBA L74 alone completely prevented LPS-mediatedeffects in mice treated with non-fermented milk, although it was lesseffective on CpG-induced CD80 upregulation. Skim milk fermented with S.thermophilus alone prevented LPS and CpG effects and reduced CD80expression. Finally, administration of skim milk fermented with bothstrains prevented LPS- and CpG-induced CD80 up-regulation and maintainedCD80 expression at basal levels. Data in FIG. 18 are expressed asmean±S.E. of three separate experiments.

Example 11: Effect of Milk Fermented by L paracasei CBA L74 onT-Lymphocyte Phenotype in the Intestinal Mucosa

Responses of intestinal lymphocytes CD4+ e CD8+ exposed to PHA. We askedwhether dietary supplementation with milk (either fermented orunfermented) would affect intestinal T-lymphocytes (CD4+ and CD8+)polarization toward a Th1 or Th2 phenotype. Peyer's Patches derivedlymphocytes were exposed to PHA, and then lymphocyte polarization wasevaluated by staining for intracellular IL-4 and IFN-γ.

As shown in FIG. 19, exposure to PHA significantly increased IL4 andIFNγ production in CD4+ lymphocytes from control mice (° means p<0.05 vsunstimulated lymphocytes from control mice). This induction was notobserved in mice treated with either non-fermented or fermented milk.Following supplementation with non-fermented milk, CD4+ lymphocytesshowed increased basal levels of IL4 and IFN-γ as compared to controlanimals (° means p<0.05 vs un-stimulated lymphocytes from control mice).These data suggested that supplementation with fermented milk preventednon-specific activation and maintained IL4 and IFN-γ production at basallevels.

As shown in FIG. 20, exposure to PHA in CD8+ intestinal lymphocytes fromcontrol mice increased production of IL4 and IFNγ (* means p<0.05 vsnon-stimulated lymphocytes from control mice). This induction was notobserved in mice treated with either non-fermented or fermented milk.Administration of milk fermented with L. paracasei and S. thermophilusat higher dose decreased the basal production of IL4 whereas followingstimulation with PHA, the levels of both IL4 and IFNγ were reduced ascompared to stimulated lymphocytes from control mice (§ means p<0.05).

Example 12: Effect of Milk Fermented by L paracasei CBA L74 onIntestinal Mucosa Histology

To determine the distribution of proliferating cells in the intestinalmucosa, we performed an immunohistochemcal analysis of expression ofKi67, an antigen expressed by dividing cells. In control mice there wasa clear immunoreactivity in the intestinal crypts, whereas there was nostaining on the villi. In the tissue from mice receiving non-fermentedmilk there was a stronger expression in the crypts, although there wasno ectopic expression of the antigen. In mice receiving the differentkind of fermented milk we observed comparable staining patterns. We haveperformed a histological evaluation of ileal mucosa of the differentexperimental groups. Results of this study are shown in FIG. 21.Administration of non-fermented milk reduced the number and length ofintestinal villi, whereas the intestinal morphology was preserved in theanimals receiving the various forms of fermented milk.

Example 13: Effect of Rice Fermented by L paracasei CBA L74 on ImmuneSystem Markers in the Intestinal Mucosa

These experiments were designed to evaluate the immuno-modulatoryproperties of L. paracasei CBA-L74-fermented cereals. Studies includeddaily intragastric administration for two weeks of: 1) Non fermentedrice; 2) Fermented rice (with L. paracasei CBA L74) 100 mg day(corresponding to 2×10⁸ cfu/L of L. paracasei CBA-L74) and 3) Fermentedrice (with L. paracasei CBA L74) 500 mg day (corresponding to 10⁹ cfu/Lof L. paracasei CBA-L74) Mice during the treatment period did not showany sign of illnesses or diarrhea. At the end of the treatment mice werekilled and the Peyer's patches (PP) were collected aseptically.Epithelial cells were removed from PP by incubation in HBSS with no Ca₂+plus EDTA and then tissues were subjected to enzymatic digestion. Thesingle cell suspension was washed, resuspended in cold RMPI and used forthe following tests.

We initially examined the effect of different diet supplements on thewhole intestinal mucosa. First, we performed haematoxylin and eosinstaining of paraffin embedded ileal sections. None of the dietsupplements used had significant effects on intestinal architecture orcaused an infiltrate of inflammatory cells.

We then determined whether administration of L. paracasei CBA L74fermented rice affected the level of anti- and pro-inflammatorycytokines in the intestinal mucosa. As shown in FIG. 22, administrationof rice or fermented rice increased both mucosal IL-1β. and IL-4.Supplementation with fermented rice at the highest dose caused areduction in IL-10 as shown in FIG. 23.

We then determined the effect of different regimen supplementation onmucosal IgA level. Following two weeks of diet supplementation, animalswere sacrificed and the intestinal mucosa collected and homogenized.Total IgA was measured by ELISA and values normalized to total mucosalproteins. None of the supplements had significant effects on mucosalIgA.

We analyzed the effects of rice or L. paracasei CBA L74-fermented riceon intestinal mucosa innate immunity. As shown in FIG. 24, dietsupplementation with rice and fermented rice (low dose) had onlymoderate effects on mucosal TLRs. As shown in FIG. 25A, dietsupplementation with non-fermented rice drastically increased mucosalPPARγ levels. High doses of fermented rice also increased mucosal PPARγlevels. As shown in FIG. 25B, two weeks of diet supplementation withnon-fermented rice reduced NF-κB activation and increased IKB in themucosa. Supplementation with fermented rice at low doses had no effectcompared to control, whereas the higher dose had effects comparable tothose observed with non-fermented rice.

We also measured the effects of the dietary administration on anti- andpro-inflammatory cytokines level in the serum. Dietary supplementationwith either rice or fermented rice (low dose) had no influence on serumIL-1β and IL-4 levels.

Example 14: Effect of Rice Fermented by L. paracasei CBA L74 onDendritic Cell Phenotype in the Intestinal Mucosa

We next focused on the impact of dietary supplementation withnonfermented rice or rice fermented with L. paracasei CBA L74 on immunecells relevant to the activity of mucosal-associated immune system,namely dendritic cells and lymphocytes. First we labeled the singlecells suspension with anti-CD1 (to identify DC) and with either antiCD-80 and CD-86, MHC-II and CD-40 to determine the level of activity andmaturity of intestinal DC in these lymphoid organs. As shown in thetable in FIG. 26, we observed a significant effect of fermented rice athigh dose on DCs phenotype. Non-fermented rice supplementation (in theseexperiments we used two different doses) had no effect, whereas theeffects of fermented rice on CD80, CD40 and MHC-II level were evident at100 mg/day and more pronounced at 500 mg/day.

We next determined the ability y of diet supplementation with rice or L.paracasei CBA L74 fermented rice to modify the reactivity of DCs topro-inflammatory stimuli (such as bacterial LPS and CpG). As shown inthe table in FIG. 27, exposure of DCs from control mice to LPS or CpGinduced an up-regulation of CD80 in control DCs. Supplementation withnon-fermented rice did not modify the reactivity to inflammatorystimuli. Supplementation with the tested dose of fermented rice reducedLPS- and CpG-induced CD80 up-regulation.

Example 15: Effect of Rice Fermented by L paracasei CBA L74 onT-Lymphocyte Phenotype in the Intestinal Mucosa

We investigated whether dietary supplementation with rice or L.paracasei CBA L74 fermented rice was able to influence intestinalT-lymphocyte (either CD4+ and CD8+) polarization toward a Th1 or Th2phenotype. Peyer's Patches derived lymphocytes were exposed to PHA andthen lymphocyte polarization was evaluated by intrakine staining forIL-4 and IFN-γ.

The results of this experiment for CD4+ and CD8+ lymphocytes are shownin FIGS. 28 and 29, respectively. As shown in FIG. 28, in the basalcondition, in CD4+ lymphocytes IL-4 and IFNγ were almost in equilibriumsince about 10-12% of the cells are positive for these cytokines. Asshown in FIG. 29, in CD8+, in the basal condition there was a slightpredominance of IL-4 over IFNγ expressing cells. Exposure of either CD4+or CD8+ lymphocytes to PHA caused a strong increase in intracellularstaining for IL-4 and IFNγ, with a predominance of IFNγ production.

In mice receiving non-fermented rice we observed a preponderance of IL-4production (Th2 phenotype) either in basal condition and following PHAstimulation for both CD4+ and CD8+ lymphocytes. However, IL-4 productionwas associated to a persistence of IFNγ production in basal conditionand, following PHA stimulation we observed an increased expression ofthis cytokine although the response was blunted as compared to theresponse in control cells. In mice receiving the fermented rice in basalcondition we observed a preponderance of IFNγ positive cells over IL-4,although in basal condition the percentage was comparable (for CD4+) orslightly lower (for CD8+) as compared to controls. Following PHAstimulation, in both cellular populations the cytokine response wasblunted as compared to control mice, although directed toward a Th1phenotype (preponderance of IFNγ positive cells).

Example 16: Effect of L paracasei CBA L74 on IL-10 Production inMonocytederived Dendritic Cells (MoDCs) in the Presence of Salmonellatyphimurium

We tested the ability of both L. paracasei CBA L74 cells and L.paracasei CBA L74 cell supernatants to induce production of theanti-inflammatory cytokine, IL-10, in human MoDCs in the presence of thebacterial pathogen, Salmonella typhimurium. Human MoDCs were obtainedfrom at least four, and in some cases, seven donors. As shown in FIG.30, Salmonella typhimurium (“FB62”), the control Lactobacillus strain,L. paracasei 21060 (“B21060”) and L. paracasei CBA L74 (“CBAL74”) cellsall induced IL-10 production. In contrast, although supernatant fromSalmonella typhimurium (“sn fb62”) induced IL-10 production,supernatants from both Lactobacilli, Lactobacillus strain, L. paracasei21060 (“sn b21060”) and L. paracasei CBA L74 (“sn cba174”) did not.Co-incubation of L. paracasei CBA L74 supernatant with Salmonellatyphimurium (“sn cba174+FB62”) induced IL-10 to levels over and abovethat seen with Salmonella typhimurium alone (“sn fb62+FB62”).Interestingly, a similar effect was observed even if the MoDCs werepreconditioned with L. paracasei CBA L74 supernatant for only one hourand then washed to remove the supernatant (“1 h sn cba174+FB62”).

Example 17: Effect of L paracasei CBA L74 on IL-12p70 Production inMonocytederived Dendritic Cells (MoDCs) in the Presence of Salmonellatyphimurium

We tested the ability of both L. paracasei CBA L74 cells and L.paracasei CBA L74 cell supernatants to induce production of thepro-inflammatory cytokine, IL-12p70, in human MoDCs in the presence ofthe bacterial pathogen, Salmonella typhimurium. Human MoDCs wereobtained from at least four, and in some cases, seven donors. Theresults of this experiment are shown in FIG. 31. In contrast to theresults obtained in Example 16 for the anti-inflammatory cytokine, wefound that Salmonella typhimurium (“FB62”) induced high levels ofIL-12p70 while the control Lactobacillus strain, L. paracasei 21060(“B21060”) and L. paracasei CBA L74 (“CBAL74”) cells induced only lowlevels of IL-12p70. Here again, supernatant from Salmonella typhimurium(“sn fb62”) induced IL-12p70 production, but supernatants from bothLactobacillus strains, L. paracasei 21060 (“sn b21060”) and L. paracaseiCBA L74 (“sn cba174”) did not. Co-incubation of L. paracasei CBA L74supernatant with Salmonella typhimurium (“sn cba174+FB62”) caused astriking reduction in IL-12p70 production. The effect was observed evenif the MoDCs were preconditioned with L. paracasei CBA L74 supernatantfor only one hour and then washed to remove the supernatant (“1 h sncba174+FB62”). This reduction exceeded that observed for the controlLactobacillus, L. paracasei 21060 (“1 h sn b21060+FB62”). These datasuggest that both L. paracasei CBA L74 and culture supernatant from L.paracasei CBA L74 have anti-inflammatory properties that can mitigatethe inflammation induced by the bacterial pathogen, Salmonellatyphimurium.

Example 18: Effect of L paracasei CBA L74 Fermented Milk on IL-10Production in Monocyte-Derived Dendritic Cells (MoDCs) in the Presenceof Salmonella typhimurium

We tested the ability of L. paracasei CBA L74-fermented milk to induceproduction of the anti-inflammatory cytokine, IL-10, in human MoDCs inthe presence of the bacterial pathogen, Salmonella typhimurium. Theresults of this experiment are shown in FIG. 32. Although unactivatedMoDCs do not usually produce IL-10, we found that incubation of MoDCswith L. paracasei CBA L74-fermented milk induced dose-dependent IL-10production (see “matrice CBAL74 1.41 gr/100 ml” and “matrice CBAL74 0.14gr/100 ml”). The capacity was retained even in the presence ofSalmonella typhimurium (see “matrice CBAL74 1.41 gr/100 ml+FB62” and“matrice CBAL74 0.14 gr/100 ml+FB62”).

FIG. 32 also shows that L. paracasei CBA L74 that had been activated byheat (“6.3*106 CFU CBA74 boiled”), paraformaldehyde treatment (“6.3*106CFU CBA74 PFA”) or freeze-thawing (“6.3*106 CFU CBA74 F&ampT”) retainedthe ability to induce IL-10.

As we observed for L. paracasei CBA L74 culture media, L. paracasei CBAL74-fermented milk caused a significant, dose-dependent reduction inproduction of IL-12p70 induced by Salmonella typhimurium. (FIG. 33). S.thermophilus fermented milk caused an increase in IL-12p70 production inthe presence of Salmonella typhimurium. However, milk fermented by L.paracasei CBA L74 did not stimulate IL-12p70 production, consistent withthe anti-inflammatory properties of L. paracasei CBA L74. These datasuggest that L. paracasei CBA L74 retained its anti-inflammatoryproperties even in the presence of more pro-inflammatory species.

Example 19: Effect of L. paracasei CBA L74 Cell Supernatants on IL-10and IL 12p70 Production in Monocyte-Derived Dendritic Cells (MoDCs) inthe Presence of Enterobacter sakazaki

We tested the ability of L. paracasei CBA L74 cell supernatants toinduce production of the anti-inflammatory cytokine, IL-10 and thepro-inflammatory cytokine, IL-12p70, in human MoDCs in the presence ofthe bacterial pathogen, Enterobacter sakazaki. We tested two strains ofE. sakazaki, N9 and N13. E. sakazaki induced the production of bothIL-10 and IL-12p70 in human MoDCs. The addition of L. paracasei CBA L74cell supernatants resulted in an increase in IL-10 and a significantdecrease in IL-12p70.

Example 20: Effect of L paracasei CBA L74 Fermented Rice on CytokineProduction in a Tissue Explant Model in the Presence of Salmonellatyphimurium

Mouse gut epithelium as used to set up three-dimensional co-culturesystem as described in Abud (Exp. Cell Res. 303: 252-262 (2005)). Tissueexplants were cultured for 24 hour in the presence of 100% 02 with apressure of one atmosphere. The lower chamber contained 1 ml of hEC DMEMplus ITS-X and EGF. The upper chamber contained 200 ml of medium pluseither S. typhimurium, L. paracasei CBA L74 fermented rice, or acombination of S. typhimurium and L. paracasei CBA L74 fermented rice.Supernatants were harvested and levels of IL-1β, TNF-α, and IL-10 wereassayed by ELISA. As shown in FIG. 34, the addition of fermented ricesignificantly reduced production of the pro-inflammatory cytokines,IL-1β and TNF-α in the presence of S. typhimurium, with only a moderateeffect on the levels of the antiinflammatory cytokine, IL-10.

It is to be understood that the present invention is by no means limitedonly to the particular constructions herein disclosed and shown in thedrawings, but also comprises any modifications or equivalents within thescope of the claims.

1-37. (canceled)
 38. A method of modulating the immune system in asubject, the method comprising: a) identifying a subject in need ofimmune system modulation; b) administering to the subject an effectiveamount of a composition comprising a food product wherein the foodproduct has been fermented by probiotic bacterium, Lactobacillusparacasei CBA L74, International Depository Accession Number LMGP-24778, wherein any Lactobacillus paracasei CBA L74 cells present inthe composition are dead or have been rendered incapable of celldivision.
 39. (canceled)
 40. The method of claim 38, wherein the subjectis a human infant.
 41. A method of treating a subject at risk for adeveloping a gastrointestinal disorder, the method comprising: a)identifying a subject at risk for a gastrointestinal disorder; b)administering to the subject an effective amount of a compositioncomprising a food product wherein the food product has been fermented byprobiotic bacterium, Lactobacillus paracasei CBA L74, InternationalDepository Accession Number LMG P-24778, wherein any Lactobacillusparacasei CBA L74 cells present in the composition are dead or have beenrendered incapable of cell division.
 42. (canceled)
 43. The method ofclaim 41, wherein the gastrointestinal disorder is a mucosal immunesystem deficit, a food allergy, a disorder associated with diarrhea, abacterial or viral infection, irritable bowel syndrome, inflammatorybowel disease, Crohn's disease, celiac disease or necrotizingenterocolitis.
 44. The method of claim 43, wherein the mucosal immunesystem deficit is an immature immune system.
 45. The method of claim 41,wherein the subject is a human infant.
 46. A method of treating asubject having a gastrointestinal disorder, the method comprising: a)identifying a subject having a gastrointestinal disorder; b)administering to the subject an effective amount of a compositioncomprising a food product wherein the food product has been fermented byprobiotic bacterium, Lactobacillus paracasei CBA L74, InternationalDepository Accession Number LMG P-24778, wherein any Lactobacillusparacasei CBA L74 cells present in the composition are dead or have beenrendered incapable of cell division.
 47. (canceled)
 48. The method ofclaim 46, wherein the gastrointestinal disorder is a mucosal immunesystem deficit, a food allergy, a disorder associated with diarrhea, abacterial or viral infection, irritable bowel syndrome, inflammatorybowel disease, Crohn's disease, celiac disease or necrotizingenterocolitis.
 49. The method of claim 48, wherein the mucosal immunesystem deficit is an immature immune system.
 50. The method of claim 46,wherein the subject is a human infant.
 51. A method of treating asubject having a gastrointestinal disorder, the method comprising: a)identifying a subject having a gastrointestinal disorder; b)administering to the subject an effective amount of a pharmaceuticalcomposition comprising probiotic bacterium, Lactobacillus paracasei CBAL74, International Depository Accession Number LMG P-24778, wherein theLactobacillus paracasei CBA L74 cells present in the composition aredead or have been rendered incapable of cell division.
 52. (canceled)53. The method of claim 51, wherein the gastrointestinal disorder is amucosal immune system deficit, a food allergy, a disorder associatedwith diarrhea, a bacterial or viral infection, irritable bowel syndrome,inflammatory bowel disease, Crohn's disease, celiac disease ornecrotizing enterocolitis.
 54. The method of claim 53, wherein themucosal immune system deficit is an immature immune system.
 55. Themethod of claim 51, wherein the subject is a human infant. 56-68.(canceled)
 69. A method of treating a subject having a gastrointestinaldisorder, the method comprising: administering to the subject aneffective amount of a pharmaceutical composition comprising a culturesupernatant of probiotic bacterium, Lactobacillus paracasei CBA L74,International Depository Accession Number LMG P-24778, wherein anyLactobacillus paracasei CBA L74 cells present in the composition aredead or have been rendered incapable of cell division
 70. The method ofclaim 69, wherein the gastrointestinal disorder is a mucosal immunesystem deficit, a food allergy, a disorder associated with diarrhea, abacterial or viral infection, irritable bowel syndrome, inflammatorybowel disease, Crohn's disease, celiac disease or necrotizingenterocolitis.
 71. The method of claim 70, wherein the mucosal immunesystem deficit is an immature immune system.
 72. The method of claim 69,wherein the subject is a human infant.
 73. The method of claim 69,wherein the composition is substantially free of intact Lactobacillusparacasei CBA L74 cells
 74. The method of claim 38, wherein the foodproduct comprises from about 1×10² cfu/g to about 10¹² cfu/g of theprobiotic bacterium per gram of dry weight.
 75. The method of claim 41,wherein the food product comprises from about 1×10² cfu/g to about 10¹²cfu/g of the probiotic bacterium per gram of dry weight.
 76. The methodof claim 46, wherein the food product comprises from about 1×10² cfu/gto about 10¹² cfu/g of the probiotic bacterium per gram of dry weight.77. The method of claim 51, wherein the probiotic bacterium is in a unitdosage form of about 1×10² cfu/g to about 1×10¹² cfu/g dry weight. 78.The method of claim 69, wherein the composition comprises 0.01 to 1,000mg per kg of the culture supernatant based on the weight of the subject.